Breast Cancer Research and Treatment 68: 239–248, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Predictors of distant metastasis at presentation in breast cancer: a studyalso evaluating associations among common biological indicators

H. Bozcuk1, G. Uslu1, E. Pestereli2, M. Samur1, M. Ozdogan, S. Karaveli2, F. Sargın2, andB. Savas1

1Department of Internal medicine, Division of Medical Oncology, 2Department of Pathology, Akdeniz UniversityMedical Faculty, Antalya, Turkey

Key words: breast cancer, c-erbB-2, metastasis, multivariate analysis

Summary

Background. To investigate the correlation among some of the commonly used clinical, pathological factors andnewer biological indicators, and to identify the independent predictors of distant metastasis at presentation inpatients with breast cancer.

Methods. The pathological specimens from 73 patients with breast cancer were retrospectively evaluated byimmunohistochemistry. Data on 13 biological indicators; ER, PR, P53, c-erbB-2, PCNA, CEA, Ki-67, Vimentin,Ulex, Nm23, Cathepsin D, Factor VIII, PS2 together with clinical and pathological factors were collected.

Results. A number of highly significant correlations were found among the biological indicators studied. By lo-gistic regression analysis, the predictors of distant metastasis at presentation in univariate tests were tumor diameter,number of lymph nodes involved, P53, c-erbB-2 and grade. In multivariate analysis, tumor diameter (P = 0.042,HR: 1.88(1.02–3.44)), c-erbB-2 expression (P = 0.035, HR: 18.20 (1.23–268.66)) and grade (P = 0.010, HR:8.05(1.66–39.00)) retained their significance.

Conclusion. Our findings show that inactivation of suppressor genes, expression of oncogenes, loss of differen-tiation, augmentation of proliferative activity, metastatic potential, angiogenesis and hormone receptor status areall interrelated facets of breast cancer pathogenesis. Patients with tumors overexpressing c-erbB-2 or with biggeror higher-grade tumors probably need to be more carefully evaluated for the presence of distant metastasis, thus bebetter staged, at presentation. This may be a new reason to test c-erbB-2 routinely in all patients with breast cancerin addition to its well-known prognostic and predictive uses.

Introduction

Pathogenesis of breast cancer is complex. Geneticdefects are increasingly being described. Some ofthe genes involved affect cellular regulation and cellgrowth and some control processes like apoptosis. Oneof the best-established genes for tumorigenesis that isfound to be amplified in patients with breast cancer isc-erbB2 [1]. The function of this gene is to act as agrowth factor receptor. It is a well-known prognosticfactor in breast cancer and is associated with favorableand unfavorable response to certain chemotherapeut-ics and hormonal manipulation [2–4]. P53, ‘guardian

of genome’, is another established breast cancer pro-gression gene that regulates the cell cycle and DNArepair. The mutation of P53 is associated with ge-netic instability [5]. Nm23 is a third gene, which isdownregulated in metastatic breast cancer [6].

Apart from progression and suppressor genes,some of which have clearly been shown to possess pro-gnostic value, a number of other molecules, are alsobiologically important in initiation and progression ofbreast cancer. One such molecule is Proliferating CellNuclear Antigen (PCNA) that binds to p21WAF1/CIP1

and inhibits progression from G1 to S phase [7].Among others are Cathepsin D, a matrix degrading

240 H Bozcuk et al.

protein which might also be another mediator of meta-stasis and has also been shown to be a marker ofpoor prognosis, PS2, an estrogen regulated protein,Factor VIII staining, a measure of microvascular dens-ity, Ulex europeaus agglutinin (UEA-1) used to showlectin binding characteristics, Ki-67 derived labelingindex that is a measure of proliferative capacity and isalso of prognostic significance, and, Vimentin whichis a mesenchymal marker [8–15].

Additionally, serum and plasma assays for estro-gen and progesterone receptors are generally availablein clinical practice and the presence of these receptorsindicates good prognosis and benefit from hormonaltherapy [16]. Classical tumor marker CEA (CarcinoEmbryonic Antigen) is also of interest.

We know that among some of the biologicallyimportant molecules and prognostic factors in breastcancer, significant correlations exist [17]. Consideringthis fact, we undertook this study to see the correl-ations among the various markers mentioned above.Furthermore, we evaluated in our cohort of Turk-ish patients, factors likely to be associated with theoccurrence of distant metastasis at presentation.

Patients and methods

We reviewed the medical notes of 73 patients whowere given a new pathological diagnosis of breast can-cer in Akdeniz University School of Medicine during1998 and 1999. Our cohort included only the patientswhose specimens were also evaluated by imunohisto-chemical methods that recently became available to us.At the same time, these patients were generally theones directly admitted to our institution rather than theones referred from another hospital in our province.

From patient records and pathology reports, wegathered information on patient characteristics, tumorfeatures and molecular markers. The specimens wereevaluated by immunohistochemistry in accordancewith the following methods.

Immunohistochemical methods

Immunohistochemical staining for the molecules men-tioned was done by using the following antibodiesfrom Dako, Glostrup, Denmark: Estrogen receptor(ER, m7047, 1:50 dilution), Progesterone receptor(PR, m3529, 1:10 dilution), P53 (m7081, 1:50 dilu-tion), c-erbB2 (A0485, 1:50 dilution), PCNA (m0879,1:50 dilution), CEA (A0115, 1:50 dilution), Ki-67

(A0047, 1:20 dilution), Vimentin (m0725, 1:25 dilu-tion), Ulex (B0279, 1:400 dilution), Nm23 (A 0095,1:50 dilution), Cathepsin-D (A0561, 1:50 dilution),PS2 (m7184, 1:40 dilution), and Factor VIII (m0616,1:100 dilution). Shortly, the biotin-streptavidin perox-idase method was applied to formalin fixed, paraffinembedded material.4 µm. sections were mounted onpoly-2-lysine coated slides, deparaffinized and hy-drated through graded alcohols to water. Optimumpretreatment and dilutions were determined by testingwith both known positive or negative material. Theslides were microwaved in citrate buffer for 10 min(min.) at 800 w microwave oven for antigen retrieval,and then allowed to cool down for 20 min. The slideswere then stained with Dako Techmate 500 automatedimmunostainer by using streptavidin-biotin method.Shortly, following endogenous peroxide and proteinblocking step, the slides were incubated with primaryantibodies for 45 min. After brief washes, incubationin a cocktail of biotinylated rabbit anti-mouse IgG/IgM (Immunoglobulin G and M) for 30 min. was per-formed. The sections were then washed and incubatedwith streptavidin-biotinylated horseradish peroxidase(HRP) complex for 30 min, reacted with diaminoben-zidine and hydrogen peroxide to visualize the end-product. Mayer’s haematoxylene was used as counterstain.

Nuclear staining for hormone receptors, Ki67,PCNA and P53, membranous staining for c-erb-B2and cytoplasmic staining for other markers were evalu-ated. Positive staining for stromal cells with CathepsinD was not considered.

For all markers, the following method of quantific-ation of immunoreactivity was utilized: This methodtook two factors into account; fraction of positivelystained cells in the sample by microscopy in highpower which varied between 0% and 100%, and in-tensity of staining, measured on a 0–4+ scale. In thisscale, 0 implied no staining, 1+; weak, 2+; moder-ate, 3+; marked, and 4+; very strong staining. Forany variable, by multiplying these two values, it waspossible to obtain a product over a denominator of400 (100 × 4(for 4+) = 400), that is the magnitudes ofexpression (the quantity of immunoreactivity) for anymarker studied ranged between 0/400 and 400/400.These ratios corresponded to the magnitude of expres-sion of various markers.

Statistical analysis

For some of the molecular markers, there was consid-erable amount of missing data. This ranged between

Predictors of metastasis in breast cancer 241

1 case (1%) for ER and PR and 51 cases (70%) forFactor VIII. In univariate tests variables were usedwithout any change. However, in the multivariate ana-lysis, missing data for the four continuous variables inthe model (tumor diameter, number of lymph nodesinvolved, c-erbB2, grade) was replaced with the meanvalue for each variable (19, 12, 3 and 14% of casesmissing, respectively).

Sperman’s rho was used to show the correlationbetween the abnormally distributed and ordinal vari-ables. To find association with distant metastasis, allvariables were first individually evaluated by univari-ate logistic regression analysis. Variables having ap value smaller than 0.20 were then entered intomultivariate logistic regression analysis in which a for-ward approach using likelihood ratio was employed.P < 0.05 was considered significant. All p valueswere two tailed. Statistical package for social sciencesrelease 9.0 (SPSS 9.0) was used for statistical analysis.

Results

The mean age was 50. The pathology was invasiveductal carcinoma in 63% of the cases. 25% had dis-tant metastasis at presentation. The majority of cases(79%) had grade II disease and in 54% of the sub-jects the tumor diameter was between 2.1 and 4.9 cm.The median number of lymph nodes involved was 2.ER was positively stained in 43% of our patients, PRin 35%, P53 in 45%, c-erbB-2 in 73%, PCNA in90%, CEA in 54%, Ki-67 in 37%, Vimentin in 13%,Ulex (UEA-1) in 64%, Nm23 in 53%, Cathepsin-D in97%, Factor VIII in 68% and PS2 in 66%. The detailsof patient characteristics and descriptive statistics formolecular markers are displayed in Table 1.

Strong positivity for c-erbB-2 (magnitude of ex-pression greater than or equal to 200/400) was seen in18 (25%) of all patients. The frequencies of metastasisaccording to magnitude of expression in c-erbB-2 neg-ative, weak positive (<200/400) and strong positive(≥200/400) subgroups were 11 (2/18), 21 (7/34) and44% (8/18), respectively.

Among the 18 variables considered, 20 signific-ant associations were found. Details can be seen inTable 2. Of note, stage was associated not only withtumor diameter (rho = 0.61, P < 0.001), and numberof lymph nodes involved (rho = 0.61, P < 0.001), butalso with P53 (rho = 0.27, P = 0.023) and c-erbB2(rho = 0.38, P = 0.001).

In the univariate tests to assess the associationwith distant metastasis, the following had a p valuesmaller than 0.20: tumor diameter, number of lymphnodes involved, P53, c-erbB2, grade, CEA andUlex. In the multivariate logistic regression analysis,tumor diameter (Wald = 4.14, P = 0.042), c-erb-B2(Wald = 4.46, P = 0.035) and grade (Wald = 6.71,P = 0.010) were independently associated with theoccurrence of distant metastasis at presentation. SeeTable 3 for details.

Discussion

As expected, we found a number of significant correl-ations among the variables in the study. To start with,P53 was expressed in 45% of the biopsies and associ-ated with the number of lymph nodes involved. This isin opposition with the study of Coppola et al., whichdid not show any association between these two vari-ables. Tumor diameter, however, was not associatedwith P53 expression and percentage expressing P53was similar to that found in the same study (50%) [17].P53 expression was also correlated with c-erbB2 andKi-67 labeling index as previously shown by Barbar-eschi et al., but not with tumor grade in contrast withthe same study [18]. Rudolph et al. had already shownthe association of P53 expression and Ki-67 labelingindex [19]. We could not find a negative associationbetween P53 and PS2 in contradiction to a previ-ous report [20]. Our findings about P53 do suggestthat genetic instability (P53 expression), proliferat-ive capacity (Ki-67 labeling index), tumor growth(c-erbB2 expression) and lymph node involvement areinter-related phenomena in breast cancer.

In this study, c-erbB2 was related with Ki-67labeling index and Ulex (UEA-1) as well. The re-lationship between c-erbB2 and Ki-67 labeling in-dex was previously shown [19]. This again supportsthat growth potential and proliferative capacities arelinked. Ki-67 labeling index also correlated withPCNA expression. This is probably so, because bothare probably good indicators of proliferative capacity.Likewise, CEA expression was associated with Ki-67labeling index, again indicating the link with prolifer-ative capacity. Ulex (UEA-1) binding correlated wellwith P53 expression. This may again imply that ge-netic instability may be associated with, or perhapscause, increased tumor vascularisation.

Cathepsin-D expression was related with the num-ber of lymph nodes involved and Factor VIII expres-

242 H Bozcuk et al.

Table 1. Patient demographics and molecular markers

General characteristics n(%) Mean (± S.D.) Min, Median, Max

Pathology (n= 72)

Ductal carcinoma 45 (63)

Other 27 (37)

Stage (n= 73)

1 10 (14)

2 36 (49)

3 9 (12)

4 18 (25)

Grade (n= 63)

1 2 (3)

2 50 (79)

3 11 (18)

Tumour diameter (n = 59)

0.1–2.0 cm 19 (32)

2.1–4.9 cm 32 (54)

5.0 cm or more 8 (14)

N number (n= 64) 3.58 (0.69) 0, 2, 24

Age (n = 72) 49.78 (1.39) 28, 48, 76

Molecular markers Positive in (%) Magnitude of expression∗ER (n = 72) 31 (43) 44.65 (8.15) 0, 0, 240

PR (n= 72) 25 (35) 17.92 (4.60) 0, 0, 200

P53 (n = 69) 31 (45) 58.57 (10.71) 0, 0, 285

c-erbB-2 (n= 71) 52 (73)∗∗ 116.61 (12.29) 0, 105, 300

PCNA (n = 59) 53 (90) 158.78 (12.14) 0, 175, 285

CEA (n = 41) 22 (54) 87.68 (14.46) 0, 20, 270

Ki67 (n = 30) 11 (37) 21.05 (9.50) 0, 0, 210

Vimentin (n = 39) 5 (13) 3.38 (2.37) 0, 0, 90

Ulex (n= 47) 30 (64) 66.49 (11.69) 0, 40, 270

Nm23 (n = 40) 21 (53) 47.58 (11.64) 0, 10, 285

Cathepsin-D (n = 61) 59 (97) 143.05 (10.19) 0, 140, 285

Factor VIII (n= 22) 15 (68) 29.32 (5.64) 0, 40, 90

PS2 (n= 41) 27 (66) 82.02 (15.91) 0, 15, 285

∗: On a 400 scale, see text for explanation.∗∗: Strong positivity for c-erb-B2 (greater than or equal to 200/400) was seen in 18(25%) of allpatients.

sion. Cathepsin-D expression may indicate the distantmetastatic potential [6]. However, in this study, wealso showed that it is linked with lymph node meta-stasis and angiogenesis. This suggests that Cathepsin-D may be involved in a spectrum of events; from localto distant spread.

PS2 expression was connected with PR and Ulexexpression. PS2 expression is thought to reflect theproliferative state of the tumour [21]. Its associationwith PR expression can be explained with that it ishormone regulated [20]. It is interesting that we failedto show a relationship between PS2 and ER expression

unlike shown by previous studies [22, 23]. This couldbe due to the hormone receptor staining technique weused or unknown factors in our study population.

Stage in our patients was associated with tumordiameter, lymph node involvement, P53 and c-erbB2expression and tumor grade. This clearly is evidenceto demonstrate that higher stage of disease at presenta-tion in breast cancer results from more aggressive bio-logical behavior and greater genetic instability of thetumor. It is attention-grabbing that ER status is posit-ively, but Vimentin staining is inversely related withage. Increased expression of ER in elderly patients

Predictors

ofmetastasis

inbreastcancer

243

Table 2. Correlation of clinicla, pathological properties and molecular markers

Number Correlation coefficient 0.225Sig. (2-tailed) 0.092N 57

Er Correlation coefficient 0.019 0.211Sig. (2-tailed) 0.886 0.097N 58 63

PR Correlation coefficient 0.180 0.139 0.559Sig. (2-tailed) 0.176 0.276 0.000N 58 63 72

P53 Correlation coefficient 0.137 0.270 0.182 0.071Sig. (2-tailed) 0.319 0.037 0.136 0.567N 55 60 68 68

c-erbB-2 Correlation coefficient 0.113 0.193 0.059 −0.105 0.380Sig. (2-tailed) 0.404 0.132 0.628 0.386 0.001N 57 62 70 70 67

PCNA Correlation coefficient 0.097 0.258 −0.084 0.029 0.078 −0.063Sig. (2-tailed) 0.518 0.068 0.530 0.830 0.565 0.643N 47 51 58 58 57 57

CEA Correlation coefficient 0.008 0.072 0.194 0.064 −0.210 0.023 0.090Sig. (2-tailed) 0.963 0.660 0.231 0.694 0.187 0.888 0.601N 35 40 40 40 41 41 36

Ki67 Correlation coefficient −0.087 0.064 0.109 0.237 0.466 0.262 0.483 0.104Sig. (2-tailed) 0.660 0.736 0.565 0.207 0.011 0.161 0.017 0.673N 28 30 30 30 29 30 24 19

Vimentin Correlation coefficient 0.087 −0.100 −0.122 −0.077 −0.291 −0.144 −0.243 −0.045 −0.126Sig. (2-tailed) 0.631 0.545 0.460 0.641 0.072 0.395 0.135 0.809 0.577N 33 39 39 39 39 37 36 31 22

Ulex Correlation coefficient −0.119 0.158 0.125 0.209 0.432 0.061 0.020 0.004 0.380 −0.211Sig. (2-tailed) 0.476 0.319 0.403 0.159 0.002 0.682 0.898 0.984 0.067 0.230N 38 42 47 47 47 47 43 34 24 34

Nm23 Correlation coefficient 0.134 −0.218 −0.296 −0.97 0.084 0.165 0.032 0.036 0.163 0.262 −0.263Sig. (2-tailed) 0.070 0.196 0.063 0.552 0.613 0.310 0.854 0.854 0.456 0.162 0.134N 34 37 40 40 39 40 36 29 23 30 34

Cath.D Correlation coefficient −0.016 0.297 0.235 −0.052 0.242 0.211 −0.124 0.058 0.007 −0.169 0.198 −0.076Sig. (2-tailed) 0.912 0.033 0.071 0.694 0.062 0.102 0.378 0.731 0.974 0.333 0.192 0.653N 47 52 60 60 60 61 53 38 26 35 45 37

Factor VIII Correlation coefficient 0.180 0.124 −0.070 0.047 0.044 −0.105 −0.015 0.086 0.273 0.085 0.204 0.250 0.649Sig. (2-tailed) 0.490 0.582 0.756 0.837 0.847 0.643 0.950 0.742 0.345 0.738 0.387 0.318 0.001N 17 22 22 22 22 22 20 17 14 18 20 18 21

244H

Bozcuk

etal.

Table 2. (continued)

PS2 Correlation coefficient 0.085 −0.165 −0.080 0.368 0.280 −0.096 0.289 −0.096 0.097 −0.077 0.461 0.052 0.222 0.033Sig. (2-tailed) 0.621 0.315 0.617 0.018 0.080 0.561 0.083 0.613 0.674 0.671 0.006 0.771 0.194 0.901N 36 39 41 41 40 39 37 30 21 33 34 34 36 17

Stage Correlation coefficient 0.606 0.605 0.164 0.100 0.273 0.380 0.040 −0.079 0.000 −0.042 −0.071 0.019 0.220 0.046 0.001Sig. (2-tailed) 0.000 0.000 0.169 0.402 0.023 0.001 0.763 0.625 1.000 0.799 0.635 0.907 0.089 0.839 0.997N 59 64 72 72 69 71 59 41 30 39 47 40 61 22 41

Age Correlation coefficient 0.016 0.008 0.337 0.044 0.152 0.054 −0.215 0.277 −0.067 −0.399 0.066 −0.266 0.219 −0.112 −0.238 −0.078Sig. (2-tailed) 0.908 0.949 0.004 0.714 0.212 0.657 0.102 0.080 0.723 0.012 0.659 0.098 0.090 0.620 0.133 0.517N 58 63 71 71 69 70 59 41 30 39 47 40 61 22 41 72

Grade Correlation coefficient 0.219 0.280 −0.079 0.012 0.207 0.260 0.004 −0.048 0.256 −0.119 −0.170 0.246 0.112 0.073 0.018 0.428 0.051Sig. (2-tailed) 0.034 0.033 0.543 0.928 0.112 0.043 0.979 0.778 0.180 0.489 0.276 0.148 0.426 0.754 0.914 0.000 0.692N 53 58 62 62 60 61 50 37 29 36 43 36 53 21 37 63 63

TDIAM Number ER PR P53 c-erbB-2 PCNA CEA Ki67 Vimentin Ulex Nm23 Cath.D Factor VIII PS2 Stage Age

Correlation coefficient is Spearman’s rho.

Predictors of metastasis in breast cancer 245

Table 3. Factors associated with distant metastasis

Univariate analysis Multivariate analysis

Parameter % with distant P Exp(B) Wald P Exp(B), 95% CI -2 Log LR P to LR

metastasis

Age∗ 0.86 1.00

<50 28

50 or older 28

Tumour diameter∗ 0.008 2.04 4.14 0.042 1,88,1.02–3.44 4.546 0.033

2 cm or less 0

>2 cm 30

LN involvement, n number∗ 0.030 1.12 0.66 0.418

0 0

1 or more 24

Pathology 0.655 0.78

Infiltrating ductal carcinoma 22

Other 27

Estrogen receptor 0.379 0.61

Negative 20

Postive 29

Progesteron receptor 0.567 1.41

Negative 26

Positive 20

P53 0.044 0.30 1.47 0.226

Negative 14

Positive 34

C-erB-2∗∗ 0.009 1.01 4.46 0.035 18,20,1.23–268.66 4.869 0.027

Negative 11

Weak positive (<200 over 400) 21

Strong positive (200 or more over 400) 44

Grade∗ 0.001 14.01 6.71 0.010 8,05,1.66–39.00 7.230 0.007

1 0

2 10

3 64

PCNA 0.702 1.28

Negative 24

Positive 20

CEA 0.086 4.62 0.52 0.473

Negative 32

Positive 9

Vimentin 0.872 1323.23

Negative 12

Positive 0

Ulex 0.058 3.15 3.68 0.055

Negative 41

Positive 18

Nm23 0.419 0.47

Negative 11

Positive 20

Cathepsin D 0.227 1.00

Negative 0

Positive 35

246 H Bozcuk et al.

Table 3. Factors associated with distant metastasis

Univariate analysis Multivariate analysis

Parameter % with distant P Exp(B) Wald P Exp(B), 95% CI -2 Log LR P to LR

metastasis

Factor VIII 0.748 0.67

Negative 14

Positive 20

PS2 0.964 0.96

Negative 14

Positive 15

Ki-67 0.919 4074.49

Negative 5

Positive 0

∗: Used as continuous variables in the analysis.∗∗: See text for the method of c-erb-B2 quantification.

Figure 1. The relationship of c-erb-B2 expression and the presence of distant metastasis at presentation.

with breast cancer is probably due to the decrease ofserum estrogen level with age and explains why hor-monal therapy is more effective in the elderly. Whetherthe age dependent change in vimentin staining holds aclinical significance remains to be explored.

Apart from the significant relationships among thefactors/features studied in this paper and their clinicalsignificances, the main motivation was to see if anyof them would be independently associated with therisk of distant metastasis when a breast cancer patientfirst presents. In this regard, the result of logistic re-

gression analysis shows that tumor diameter, c-erbB2expression and tumor grade are independently relatedwith the occurrence of distant metastasis at presenta-tion. Tumor diameter and tumor grade were previouslyproved to be independent indicators of distant meta-stasis, and so were lymph node status, Ki-67 labelingindex and PR status [19]. In our study, Ki-67 labelingindex and PR status did not correlate with distantmetastasis, but in the univariate test, lymph node statusdid. However, this disappeared in the multivariateanalysis.

Predictors of metastasis in breast cancer 247

To the best of our knowledge, this is the first timec-erbB2 expression has been shown to be an independ-ent predictor of the occurrence of distant metastasis atpresentation. C-erbB2 is an oncoprotein that was pre-viously suggested to be linked with elevated mitoticrate, poor response to chemotherapy (cyclophosph-amide, methotrexate, 5-Fluorouracil), and hormonaltherapy (Tamoxifen), poor prognosis in untreated pa-tients and modulation of metastatic capacity in tumormodels [2–4]. Similarly, positivity for c-erbB-2, butnot for P53 mutations, was recently found to be signi-ficantly associated with increased risk of relapse anddeath and decreased relapse free and overall survivalin high risk primary breast cancer patients treatedwith high dose chemotherapy [24]. We believe it isthis aggressive feature that possibly makes c-erbB-2an independent indicator of the presence of distantmetastasis at the time of presentation.

In our view, it may be better to test all breastcancer patients with seemingly non-metastatic diseaseat diagnosis for c-erbB-2 for at least two good reas-ons: firstly, the data from this limited study suggeststhat tumors that are likely to have more metastaticpotential or have spread distantly at presentation arelarger and higher-grade tumors and notably expressc-erbB-2. Thus, we feel that, these tumors need tobe more carefully staged at presentation in order toavoid unnecessary treatment. Secondly, when adjuvanttherapy is indicated, it seems that patients with c-erbB-2 positive tumors may be better treated witha doxorubicin-containing regimen, as proposed byHayes, et al [3].

Therefore, we believe our finding is in favor ofroutine testing for c-erbB-2 at the time of diagnosis inall breast cancer patients. It also raises the possibilityof a new indicator role for c-erbB-2 in estimating therisk of distant metastasis. However, further work isrequired to fully test the clinical significance of thisnew indicator role.

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